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At least 631 records · Page 35Linked to original sources
PubMed1998

Automated sample preparation for drugs in plasma using a solid-phase extraction workstation.

An automated solid-phase extraction workstation was used to develop, characterize and validate two separate HPLC methods for quantifying drugs in plasma. Method development was facilitated by workstation functions which allowed wash solvents of varying organic composition to be mixed and tested automatically. The precision estimates for the two methods were within 6.0 and 2.0% RSD across their respective calibration ranges. Accuracies for replicate determinations of quality controls were between -1.2 and +4.8 RE over ng ml-1 calibration ranges, respectively. Optimized recoveries were quantitative and were generally greater than 90% for the four analytes tested, and depended to a great extent, as expected, on the composition of the wash solvent. Sample throughput benchmarks for the two methods ranged from 3 to 10 min per sample, depending on the extent of air drying used. Because of parallel sample processing, 60 samples could be extracted in as little as 17 min.

Blood Chemical Analysis↗
PubMed2002

Investigation of propofol renal elimination by HPLC using supported liquid membrane procedure for sample preparation.

One of the least explored subjects in the research on the metabolism of a widely used anaesthetic, propofol, is its excretion in an unchanged form. According to literature, the estimated percentage of applied propofol eliminated intact via kidneys is lower than 0.3%. The present study shows the amount of propofol excreted in an unchanged form with urine collected during the first 48 h after anaesthesia in five patients undergoing elective intracranial procedures. The drug was concentrated and selectively isolated from urine samples by supported liquid membrane technique and determined by HPLC with fluorescence detection. The amount of unchanged propofol eliminated with urine was approximately (0.004 +/- 0.002)% of the total applied dose. The obtained results may suggest that propofol in an unchanged form is not excreted by kidneys at all provided that all propofol determined in presented study originated from conjugates hydrolysis.

Adult↗
PubMed2005

Sample preparation procedures for biological atomic force microscopy.

Since the late 1980s, atomic force microscopy (AFM) has been increasingly used in biological sciences and it is now established as a versatile tool to address the structure, properties and functions of biological specimens. AFM is unique in that it provides three-dimensional images of biological structures, including biomolecules, lipid films, 2D protein crystals and cells, under physiological conditions and with unprecedented resolution. A crucial prerequisite for successful, reliable biological AFM is that the samples need to be well attached to a solid substrate using appropriate, nondestructive methods. In this review, we discuss common techniques for immobilizing biological specimens for AFM studies.

Animals↗
PubMed1981

The use of bonded-phase extraction columns for rapid sample preparation of benzodiazepines and metabolites from serum for HPLC analysis.

A simple, rapid method for the determination of the benzodiazepines, diazepam and chlordiazepoxide, and their metabolites by high performance liquid chromatography (HPLC) has been developed. The procedure is applicable to the assay of other similar drugs in biological fluids. The method utilizes BondElut extraction columns to facilitate the extraction. BondElut columns selectively adsorb the benzodiazepines and metabolites from serum at a pH of 9.0. The compounds are eluted with 300 microliters of methanol which makes sample concentration rapid, if even necessary. Analysis is performed using isocratic reversed-phase chromatography, and quantitation is carried out by ultraviolet (UV) detection. Using this procedure, it is possible to determine drug and metabolite levels to as low as 25 ng/ml in 0.5 ml of serum.

Anti-Anxiety Agents↗
PubMed1991

Quantitation of tryptophan and other plasma amino acids by automated pre-column o-phthaldialdehyde derivatization high-performance liquid chromatography: improved sample preparation.

Pre-column derivatization with o-phthaldialdehyde is a rapid and sensitive method for the quantitation of amino acids in biological fluids. This method uses acetonitrile as a deproteinizing reagent which gives improved recovery of tryptophan compared with 5-sulfosalicylic acid and permits the measurement of aspartic acid which coelutes with 5-sulfosalicylic acid. The method is automated to increase reproducibility and convenience. Mean coefficients of variation for peak areas relative to internal standard were 3.2 and 5.2% for amino acid standards and plasma samples, respectively. The presence of nitrilotriacetic acid stabilized the o-phthaldialdehyde reagent which is important in an automated system. The method is suitable for the analysis of large numbers of plasma samples where total tryptophan and aspartic acid are of interest.

Acetonitriles↗
PubMed2001

Alkyl diol silica: restricted access pre-column packings for fast liquid chromatography-integrated sample preparation of biological fluids.

A silica-based pre-column packed with a 'restricted access material' was used for fully automated solid-phase extraction of drugs and metabolites from untreated biological fluids via a simple valve-switching method. The bifunctional sorbent employed, alkyl diol silica, is characterised by a different chemical modification of the outer surface (diol groups) and the pore surface of the particles (alkyl chains: C4, C8 or C18). This allows for a fast and efficient clean-up of complex biological samples, such as blood, serum, urine or milk, with exclusion of the macromolecular matrix (proteins, nucleic acids) in less than 1.5 min.

Animals↗
PubMed2000

Determination of albendazole and its main metabolites in ovine plasma by liquid chromatography with dialysis as an integrated sample preparation technique.

Albendazole is a benzimidazole derivative with a broad-spectrum activity against human and animal helminth parasites. In order to determine the main pharmacokinetic parameters in sheep after oral and intravenous administration of a new formulation of albendazole (an aqueous solution), a fully automated method was developed for the determination of this drug and its main metabolites, albendazole sulfoxide (active metabolite) and sulfone in ovine plasma. This method involves dialysis as purification step, followed by enrichment of the dialysate on a precolumn and liquid chromatography (LC). All sample handling operations were executed automatically by means of an ASTED XL system. After conditioning of the trace enrichment column (TEC) packed with octadecyl silica with pH 6.0 phosphate buffer containing sodium azide, the plasma sample, in which a protein releasing reagent (1 M HCl) containing Triton X-100 was automatically added, was loaded in the donor channel and dialysed on a cellulose acetate membrane in the static-pulsed mode. The dialysis liquid consisted of pH 2.5 phosphate buffer. By rotation of a switching valve, the analytes were eluted from the TEC in the back-flush mode by the LC mobile phase and transferred to the analytical column, packed with octyl silica. The chromatographic separation was performed at 35 degrees C and the analytes were monitored photometrically at 295 nm. Due to the differences in hydrophobic character between albendazole and its metabolites, a gradient elution was applied. The mobile phase consisted of a mixture of acetonitrile and pH 6.0 phosphate buffer. The proportion of organic modifier was increased from 10.0 to 50.1% in 12.30 min, then from 50.1 to 66.9% in 1.70 min. First, the gradient conditions and the temperature were optimised for the LC separation using the DryLab software. Then, the influence of some parameters of the dialysis process on analyte recovery was investigated. Finally, the method developed was validated. The mean recoveries for albendazole and its metabolites were about 70 and 65%, respectively. The limits of quantification for albendazole and its metabolites were 10 and 7.5 ng/ml, respectively.

Albendazole↗
PubMed1988

Liquid chromatographic analysis of doxazosin in human serum with manual and robotic sample preparation.

A specific method for the determination of the antihypertensive drug doxazosin in human serum is described. The method utilizes the related drug prazosin as an internal standard and is based on a simple extraction scheme followed by analysis by reversed-phase ion suppression high-performance liquid chromatography (HPLC) on an alumina-based column with fluorescence detection. The method is completely automated with a flexible robotic system for the analysis of drugs in biological fluids. The robotic automation of the method allows a 20% increase in the sample throughput and the savings of about 7 man-hours a day. Both the manual and robotic procedures yield precise quantitative results over the therapeutically relevant concentration range of 0.5 to 20 ng/mL of serum.

Chemical Phenomena↗
PubMed2000

Alpha spectrometry sample preparation using selectively adsorbing thin films

Several years ago, Switzerland introduced limits for natural radionuclides in food, e.g. 1 Bq/l for 226Ra or 10 Bq/l for the sum of 238U and 234U in drinking water. To make enforcement by regional (cantonal) laboratories more attractive, simplified analytical methods had to be offered, at least for drinking water. A first step has been the development of radium adsorbing sheets. A 20 mm x 20 mm MnO2 film on a polyamide substrate adsorbs more than 80% of the radium present in a 100 ml water sample within 6 h. The film is thin enough to allow for high resolution alpha spectrometry. A second step now under way is to produce thin films, which selectively adsorb uranium. Actually, an ion exchange resin with diphosphonic and sulfonic acid groups is used for this purpose. Although not yet very thin, these films make possible energy resolutions far better than with any liquid scintillation alpha spectrometry method. Adsorption efficiencies are more than 80% after 20 h exposition to a 100 ml water sample (20 mm x 20 mm sheet). A third step is to have a system that measures radionuclide concentrations in water on-line. A prototype is presented where radionuclides are adsorbed on a film in contact with the water. A Si-detector placed on the other side of the film support counts the alphas passing through.

Journal Article↗
PubMed1998

High-performance liquid chromatography tandem mass spectrometry procedure with automated solid phase extraction sample preparation for the quantitative determination of paclitaxel (Taxol) in human plasma.

A sensitive, specific, accurate and reproducible analytical method was developed and validated for the quantitation of the anticancer agent paclitaxel in human plasma. This procedure is based on high performance liquid chromatography/ion spray-tandem mass spectrometry. This methodology is highly specific because a MS/MS technique (multiple reactant-ion monitoring, MRM) was used for both paclitaxel and its internal standard. The use of a fully automated solid phase extraction procedure, using a CN Sep-pak cartridge, to improve the detection limit and quantification limit of paclitaxel in human plasma samples, was evaluated. The method involves the addition of methyl-paclitaxel as internal standard (i.s.). The retention times of paclitaxel and the I.S. were 2.8 and 4.0 min., respectively. The assay was linear over the range 5 to 500 ng/mL, with a quantification limit of 5 ng/mL having a coefficient of variation (c.v.) < 10%. Standard calibration curves, performed on three different days, had correlation coefficients always greater than 0.998. The intra and inter-day precision were within 12%, and accuracy was included in the range 102-110%. Paclitaxel recovery assessed at 15,250 and 500 ng/mL, was determined to be greater than 85%. The assay is applicable to clinical pharmacokinetic studies.

Antineoplastic Agents, Phytogenic↗
PubMed1986

Sample preparation for inositol measurement: Sep-Pak C18 use in detergent removal.

The enzymatic-fluorometric method of myo-inositol quantitation by L.C. MacGregor and F.M. Matschinsky (1984, Anal. Biochem. 141, 382-389) has proved to be very useful in accurately measuring small amounts of myo-inositol. Although this procedure is quite satisfactory, relatively high concentrations of detergents, salts, NADH, and malate interfere. To extend the usefulness of the MacGregor and Matschinsky method we report here an extraction procedure which removed the interfering substances, yet allowed the recovery of close to 100% of the inositol. The procedure involved first passing the sample through a Sep-Pak C18 cartridge to remove detergent and then through a mixed-bed resin to remove the ionic constituents. The procedure with the Sep-Pak C18 cartridge is applicable to a wide variety of biological samples requiring detergent removal.

Animals↗
PubMed2005

Real-time PCR quantitation of hepatitis B virus DNA using automated sample preparation and murine cytomegalovirus internal control.

Quantitation of circulating hepatitis B virus (HBV) DNA is important for monitoring disease progression and for assessing the response to antiviral therapy. Several commercial and 'in house' assays for HBV DNA quantitation have been described but many of these have limitations of relatively low sensitivity and limited dynamic range. This study describes the development and evaluation of a FRET-based real-time PCR assay designed to overcome these limitations and to provide accurate quantitation of DNA from all eight genotypes of HBV (A-H). The assay employs a fully automated nucleic acid extraction system permitting high-sample throughput with minimal 'hands-on' time and incorporates a murine cytomegalovirus (mCMV) internal control to prevent false negative results and under-reporting due to unrecognised problems with viral lysis, DNA purification or PCR amplification. Sensitivity, assessed by Probit analysis at the 95% detection level, was 24.4 IU/ml, associated with an extremely wide dynamic range (approximately 9 log10). Coefficients of variation were low for both intra-assay and inter-assay variability (CV%, 7-11%) and quantitative data correlated well (R2 = 0.97) with the Digene hybrid capture assay. This assay provides an ideal system for therapeutic monitoring and for studying the relationship between HBV viral load and stage of disease.

DNA, Viral↗
PubMed2000

The advantages of cell lysis before blood sample preparation by extraction for HPLC propofol analysis.

Propofol (2,6-diisopropylphenol) is a short-acting drug with a large volume of distribution and high body clearance. It is suitable both for the induction of anaesthesia by bolus injection and the maintenance of anaesthesia by repeated injections or a continuous infusion. Examining the drug concentration its analysis in whole blood is recommended. This results from the fact that propofol molecules strongly bind with plasma proteins and cellular blood constituents and blood composition variations are observed between individuals or in different disease states or resulting from transfusion etc. In most cases the HPLC analysis follows the extraction of samples. The degree of propofol binding with blood cells can be different, depending on the blood type, and it can change in time, which may affect the results of the analysis. The paper discusses and shows the necessity of blood cell lysis before the extraction procedure. The cell lysis makes possible to determine the total amount of propofol in blood independently of the degree of propofol binding with cellular blood constituents and its changes.

Adult↗
PubMed1999

Simplified sample preparation for the analysis of oligonucleotides by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry.

Matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOFMS) for the analyses of oligonucleotides has generally been carried out using negative ionisation conditions, usually following ammonium ion-exchange chromatography and the addition of ammonium buffers to the MALDI matrix. The molecular ion region is complex, due to the varying degrees of ammoniation of the phosphate backbone of the oligonucleotide. This gives rise to an overall decrease in sensitivity compared with similar size peptides and can cause ambiguity of assignment of the relative molecular mass of the sample. This study describes the use of H(+) ion exchange resin in situ as the means of removing alkali metal ions from the phosphate backbone of the oligonucleotide. An increase in resolution, sensitivity and identification of the molecular species is reported, with little or no difference in sensitivity observed between positive or negative ionisation spectra. This method is now used for routine screening of synthetic oligonucleotides with a gain in sensitivity of 1-2 orders of magnitude compared with previous methods, and mass assignment errors of +/-0.1% are routinely recorded for externally calibrated data.

Ion Exchange Resins↗
PubMed1993

Sample preparation for plasma measurement of plasminogen activator inhibitor-1 antigen in large population studies.

Plasminogen activator inhibitor-1 is important in regulating fibrinolysis and may be an important cardiovascular risk factor. Because of this, there is increased interest in performing plasminogen activator inhibitor-1 assays in large epidemiologic studies. Our aim in this study was to determine the simplest blood collection methods that yield accurate results with our plasminogen activator inhibitor-1 antigen assay. Our results indicate that the following issues are important: (1) since there is a large circadian variation in plasminogen activator inhibitor-1 plasma levels, a target time frame must be established; (2) commercially available citrate collection tubes are adequate, if sample processing is rapid; (3) careful venipuncture is necessary, with freely flowing collection; hemolysis must be avoided; and (4) centrifugation of at least 30,000 g.min is required to avoid platelet contamination.

Blood Specimen Collection↗
PubMed1997

Automated sample preparation using membrane microtiter extraction for bioanalytical mass spectrometry.

The development and application of membrane solid phase extraction (SPE) in 96-well microtiter plate format is described for the automated analysis of drugs in biological fluids. The small bed volume of the membrane allows elution of the analyte in a very small solvent volume, permitting direct HPLC injection and negating the need for the time consuming solvent evaporation step. A programmable liquid handling station (Quadra 96) was modified to automate all SPE steps. To avoid drying of the SPE bed and to enhance the analytical precision a novel protocol for performing the condition, load and wash steps in rapid succession was utilized. A block of 96 samples can now be extracted in 10 min., about 30 times faster than manual solvent extraction or single cartridge SPE methods. This processing speed complements the high-throughput speed of contemporary high performance liquid chromatography mass spectrometry (HPLC/MS) analysis. The quantitative analysis of a test analyte (Ziprasidone) in plasma demonstrates the utility and throughput of membrane SPE in combination with HPLC/MS. The results obtained with the current automated procedure compare favorably with those obtained using solvent and traditional solid phase extraction methods. The method has been used for the analysis of numerous drug prototypes in biological fluids to support drug discovery efforts.

Autoanalysis↗
PubMed1996

Use of aqueous two-phase systems in sample preparation for polymerase chain reaction-based detection of microorganisms.

An aqueous two-phase system, consisting of poly(ethylene glycol) (PEG) and dextran, was employed to separate polymerase chain reaction (PCR)-inhibitory substances from bacterial cells. The PCR inhibition of four soft cheeses was examined and three of them were found to be strongly PCR-inhibitory. Extraction of the PCR-inhibitory soft cheeses inoculated with Listeria monocytogenes in an aqueous two-phase system containing 8% (w/w) PEG 4000 and 8% (w/w) dextran 500, was found to lower the PCR detection level of L. monocytogenes by more than four orders of magnitude in two of the cheeses compared to the case where no such sample pretreatment was performed. Depending on the type of cheese used, the PCR-inhibitory factors were found to be enriched in either the top or bottom phase in the aqueous two-phase system. These results show that different soft cheeses contain different types and amounts of PCR-inhibitory substances.

Cheese↗